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如何利用基因魔剪—CRISPR技术来进行非编码基因组功能的研究?

2016-10-23 佚名 生物谷

我们或许才刚刚开始打开对巨大的基因组非编码区域的研究,基于CRISPR的两项新技术或许就能够帮我们开启研究的新篇章。我们(原文笔者)能够很好地理解基因组中编码蛋白组分背后的规则,同时通过对DNA序列的观察我们也能够找出从事编码作用的基因的开始以及终止位置。 对于基因组中残留的98%的基因组而言,却又是一番不同的故事了,如今我们对于DNA暗物质的理解都是来自于对非编码DNA单个

我们或许才刚刚开始打开对巨大的基因组非编码区域的研究,基于CRISPR的两项新技术或许就能够帮我们开启研究的新篇章。我们(原文笔者)能够很好地理解基因组中编码蛋白组分背后的规则,同时通过对DNA序列的观察我们也能够找出从事编码作用的基因的开始以及终止位置。

对于基因组中残留的98%的基因组而言,却又是一番不同的故事了,如今我们对于DNA暗物质的理解都是来自于对非编码DNA单个片段的研究和理解,而真正指导非编码基因组发挥作用的规则目前研究者并不清楚。博德研究所的创始主任Eric Lander说道,90%的影响人类疾病的遗传突变都位于非编码区域,但如今我们并没有有效的方法去阐明到底是哪些调节子在影响着这些基因的表达

刊登在Science杂志上的一篇研究报告中,来自博德研究所的两个研究团队通过研究利用CRISPR基因编辑技术揭示了非编码基因工作的机制。利用两种互补的方法,研究人员以CRISPR作为工具系统性地同时调查了数千个非编码的DNA序列,研究人员(来自Lander实验室和来自Zhang Feng实验室的研究者)在研究中鉴别出了多个非常有意思的遗传调节子,其中就包括那些远离所控制基因位点的数以百万计的碱基。

研究者Jesse Engreitz指出,我们希望能够在每个细胞周期中对控制每个基因表达的非编码元件进行编录,这在生物学研究上是一个非常大的问题,而且这也是一个限速的步骤,即将和基础分子机制相关的许多遗传特性同人类疾病相联系。

变化多端

目前两个研究小组都利用了CRISPR进行了相关筛查来干扰非编码的DNA,但两个研究小组却是利用不同的方法来进行了相关研究。研究者张峰(Zhang Feng)利用Cas9来对非编码DNA的重叠延伸片段进行精准的切割,在这种情况下,围绕在三个基因NF1, NF2和CUL3周围区域的功能就会丧失,而这与某些形式的黑色素瘤耐药性产生直接相关。研究者解释道,这种方法就能够帮助我们诱导一系列多样性突变的产生,而且我们也不必推测既定的序列为何会被干扰。

另外一组研究者Engreitz等人则利用了CRISPR的干扰系统,即利用失活形式的Cas9同KRAB蛋白相互融合从而沉默靶向序列的表达,这些靶向序列围绕在MYC和GATA1附近,这两个基因是重要的转录因子。Engreitz说道,这种系统能够帮助我们队非编码区域的调节性影响进行定量的评估,同时其还能够为我们展示如何能够控制一个既定的基因。

每个研究小组都利用了一种功能性的“读数器”以及深度的测序技术观察了哪种导向RNAs能够影响目标基因的表达,同时还能够帮助绘制出导向RNAs所影响的调节子的图谱。两个研究团队的研究结果都阐明了利用CRISPR工具追踪非编码基因组调节机制的重要性。研究者Engreitz等人阐明了7个MYC和3个GATA1增强子的重要性,而张峰研究团队则筛选出了多个增强子以及仅结合CUL3的转录因子结合位点。

在“自然栖息地”对序列进行研究

类似于传统的报道分子实验,即科学家们感兴趣的在质粒中将序列同报道自偶联进行研究;这些混合的CRISPR筛选技术有着明显的不同,其能够直接探查序列,也就是说在最原始的位点进行序列的研究解读。研究者Sanjana强调道,这些筛选技术能够从内源性的角度来对序列进行研究,同时报道子实验也是非常有帮助的,但其缺少了3D构象和局部的染色质环境,在这里这些调节性序列或许就会经历正常的一些相互作用。

研究者表示,举个例子说,我们或许能够在基因启动子和非编码位点之间观察到长期的循环结构,但如果我们仅仅观察这些调节性原件的话或许就会错过一些感兴趣的3D相互作用。Engreitz指出,目前存在一种限制,不管是CRISPR方法还是别的方法,在当前的形式下,发现基因组固有的冗余程度或许并不足以破碎一种增强子来理解基因被控制机制,或许我们需要对多个增强子进行打断来研究。

但Engreitz及其同事表示非常乐观,他们希望能够利用基于CRISPR的技术来阐明非编码基因组背后所隐藏的秘密。目前在非编码基因组上研究的最大挑战就是,尽管非编码基因组非常庞大,但其中所包含的单一功能性元件却是非常小的,未来研究者希望开发新的研究方法在保证非编码基因组较高分辨率的情况下对更大的基因组区域进行探索。

研究者指出,随着他们绘制出多个图谱以及其相关性,他们相信能够帮助预测关于非编码基因组的一些信息;利用导向RNAs文库引入CRISPR或者抑制子或许就能够帮助研究者阐明大块区域的非编码DNA对不同基因的作用以及效应,而届时科学家们或许才刚刚打开对基因调节的系统性图谱研究的第一步。

参考资料:

【1】Reading the rules of gene regulation with CRISPR

【2】High-resolution interrogation of functional elements in the noncoding genome

Science   DOI: 10.1126/science.aaf7613

【3】Systematic mapping of functional enhancer-promoter connections with CRISPR interference

Science    DOI: 10.1126/science.aag2445

【4】A massive approach to finding what's "real" in genome-wide association data

【5】CRISPR system scales up in human cells

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    2017-01-13 d830384
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    2016-10-25 yuandd
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